Invasion by T. cruzi of Vero cells colonized with Coxiella burnetii
The study of cell co-infection may allow the observation of the behavior of pathogens in the presence of one another, and provide new insights onthe course of infection and interaction of each pathogen with the endocytic pathway (Rabinovitch et al. 1999, Rabinovitch and Veras 1996). In the last years, we began to examine the behavior of T. cruzi trypomastigotes upon invasion of cells that had been previously colonized with Coxiella burnetii, an obligate intracellular bacterium and causative agent of Q fever, an opportunistic human pneumonia. C. burnetii may inhabit both phagocytic and non-phagocytic cells (Baca and Paretsky 1983) where it forms large cytoplasmic vacuoles with lysosomal characteristics by acquisition of hydrolases and lysosomal markers (LAMP-1 and LAMP-2). C. burnetii is a well adapted organism that accomplishes all metabolic processes at low pH (Hackstadt and Williams 1981), as it has been established that their vacuoles maintain an acidic pH during infection (Maurin et al. 1992). A previously demonstrated hallmark of C. burnetii vacuoles is their fusogenicity: from inert particles to different intracellular pathogens can easily be targeted to this new compartment (Rabinovitch et al. 1999). A very useful quality of this system is that persistent infection can be easily established and cells harboring large C. burnetti vacuoles can be maintained in culture for several weeks. We began to exploit this feature to examine the co-infection with T. cruzi.
TRYPOMASTIGOTES
The presence of the bacterium (in persistent infections) per se can hinder infection by trypomastigotes (TCTs and metacyclics, CL strain) in Vero cells. However, inhibitors of vacuolar ATPases and weak bases that also raise intravacuolar pH have a dramatic effect on the invasion processes (Andreoli and Mortara 2003a). Whereas in Vero cells, raising pH reduces infectivity, presumably by affecting the lysosomal pathway (Andrews 1995, Tardieux et al. 1992), cells colonized with C. burnetii are more susceptible to trypomastigote invasion than the untreated controls. This unexpected effect probably reflects the fragmentation of the large vacuole when cells are treated with these drugs (Figure 5). One possibility is that LAMP-1 molecules become increasingly exposed at the cell surface thus facilitating the lysosomal route for internalization (Kima et al. 2000).
The ultimate goal of these experiments was to transfer trypomastigotes from the cytoplasm to the C. burnetii vacuole, through the fusion between the bacterium vacuole and PV. Metacyclic trypomastigotes were readily transferred but TCTs escaped from their PVs and released themselves into the cytoplasm. This difference can be accounted for by the low TcTOX and transialidase activities in metacyclics: since these forms remain longer in their PVs (Stecconi-Silva et al. 2003), they have more opportunities to be transferred to the C. burnetii vacuole (Andreoli and Mortara 2003a). Intravacuolar pH measurements in live cells indicated that trypomastigotes are preferentially transferred to more acidic vacuoles (pH 4.0-4.7), and raising vacuolar pH with the compounds mentioned above, dramatically decreased transfer efficiency (Andreoli and Mortara 2003a). A previously undescribed release of LAMP-1 from the PV is shown in figure 6. In these studies with cells transfected with GFP-LAMP-1 we have also obtained evidence that internalization of metacyclic trypomastigotes may involve erratic translocations of parasites surrounded by PV membrane through the cytoplasm (Figure 6). Comparative studies between TCTs and metacyclic trypomastigotes of T. cruzi I and T. cruzi II strains suggested, again, that trafficking in Vero cells colonized with C. burnetii may vary substantially among the different isolates and infective forms (Table I, L'Abbate C., unpublished observations).
AMASTIGOSTES
Bearing in mind our previous experience with amastigote invasion of HeLa and Vero cells, we have been examining how the intracellular bacteria persistently growing inside Vero cells could affect the process. We compared T. cruzi I (G strain) extracellular amastigotes with T. cruzi II parasites (CL strain). Unlike to what was seen for the trypomastigotes, the presence of the bacterium per se increased amastigote infectivity of parasites of both strains (Table I). The observation that the transference of amastigotes to the C. burnetii vacuole was enhanced by weak bases but reduced by vacuolar ATPase inhibitors (Fernandes M.C., unpublished observations) is a strong evidence that the intracellular trafficking compartments used by amastigotes (and possibly their maturation) are different form those used by trypomastigotes (Andreoli and Mortara 2003a).
GROWTH AND DIFFERENTIATION OF TRYPOMASTIGOTES WITHIN THE Coxiella burnetii VACUOLE
Once inside the C. burnetii vacuole, metacyclics differentiate into amastigotes as well as in epimastigote-like forms. This can be demonstrated by morphological examination and immunolabeling parasites with specific anti-amastigote (Barros et al. 1997) and anti-epimastigote (Almeida-de-Faria et al. 1999) antibodies. Intravacuolar pH measurements in vivo indicate that in spite of the acidic milieu, amastigotes retained a neutral pH in their cytoplasm while growing in the bacterium vacuole (Andreoli and Mortara 2003b). Using T. cruzi transfected with histone 2-GFP (Yamauchi et al. 1997) we confirmed that amastigote division takes about 70 min (Figure 7). In spite of several attempts, we could not demonstrate the transformation of amastigotes growing inside the C. burnetii vacuole into trypomastigotes.
Indications that cytoplasmic parasites grewsubstantially outside the vacuole in doubly-infected cells after 48-72h, without parallel infection by new trypomastigotes, led us to investigate whether amastigotes and/or epimastigotes could be escaping from the bacterium vacuole. Studies involving live cell video imaging, confocal and electron microscopy strongly suggested that these forms can escape from the bacterium vacuole (Andreoli and Mortara 2003b). We also demonstrated that amastigotes express C9-related TcTOX inside the C. burnetii vacuole that might be important for disruption of the bacterium vacuolar membrane.
Answers to all your Biology Questions
